Colorimetric and/or luminescent compounds may offer researchers the opportunity to use color and light to analyze samples, investigate reactions, and perform assays, either qualitatively or quantitatively. Generally, brighter, more photostable reporters may permit faster, more sensitive, and more selective methods to be utilized in such research.
While a colorimetric compound absorbs light, and may be detected by that absorbance, a luminescent compound, or luminophore, is a compound that emits light. A luminescence method, in turn, is a method that involves detecting light emitted by a luminophore, and using properties of that light to understand properties of the luminophore and its environment. Luminescence methods may be based on chemiluminescence and/or photoluminescence and/or sonoluminescence, among others, and may be used in spectroscopy, microscopy, immunoassays, and hybridization assays, among others.
Photoluminescence is a particular type of luminescence that involves the absorption and subsequent re-emission of light. In photoluminescence, a luminophore is excited from a low-energy ground state into a higher-energy excited state by the absorption of a photon of light. The energy associated with this transition is subsequently lost through one or more of several mechanisms, including production of a photon through fluorescence or phosphorescence.
Photoluminescence may be characterized by a number of parameters, including extinction coefficient, excitation and emission spectrum, Stokes' shift, luminescence lifetime, and quantum yield. An extinction coefficient is a wavelength-dependent measure of the absorbing power of a luminophore. An excitation spectrum is the dependence of emission intensity upon the excitation wavelength, measured at a single constant emission wavelength. An emission spectrum is the wavelength distribution of the emission, measured after excitation with a single constant excitation wavelength. A Stokes' shift is the difference in wavelengths between the maximum of the emission spectrum and the maximum of the absorption spectrum. The luminescence lifetime is the average time that a luminophore spends in the excited state prior to returning to the ground state and emission of a photon. The quantum yield is the ratio of the number of photons emitted to the number of photons absorbed by a luminophore.
Hydrogels are used in medicine as surgical implants (see e.g., Regenerative Medicine Applications in Organ Transplantation, G. Orlando, J. P. Lerut, S. Soker, R. J. Stratta (Ed.), Elsevier, 2014), including prevention of heart aneurysms during the post-infarction period (N. Landa et al. Effect of injectable alginate implant on cardiac re-modeling and function after recent and old infarcts in rat. Circulation, 2008, 117 (11), 1388-1396), as fillers to eliminate defects in bones and as alginate dressings for plugging deep infected wounds. Hydrogels are also used in clinical pharmacology as transport systems for the targeted delivery of drugs (Polyethylene Glycols—Advances in Research and Application. Ed. Q. A. Acton. ScholarlyEditions, Atlanta, Ga., 2013) as well as in many other medical, veterinary, biological and pharmaceutical applications (Progress in Molecular and Environmental Bioengineering—from Analysis and Modeling to Technology Applications. A. Carpi (Ed.). Chapter 5. S. K. H. Gulrez et al., Hydrogels: Methods of Preparation, Characterisation and Applications. InTech, 2011, 660. ISBN 978-953-307-268-5, DOI: 10.5772/771).
In all these applications, an important task is the visualization and monitoring of hydrogels and/or their biodegradation products (decomposition in biological tissues), in particular in real time, as well as determining the rheological properties of hydrogels, i.e. their mechanical resistance and viscoelastic characteristics.
Luminescent methods are commonly used for the detection of hydrogels in biological tissues in vivo as these methods are more sensitive compared to non-fluorescent methods as they only require 10−6-10−9M concentrations of a luminescent reporter.
A current luminescence-based, non-invasive method for in-vivo monitoring of biodegradable gelatin hydrogels is based on the luminescence of a meso-brominated pentamethine cyanine dyes (E. A. Owens et al. Highly Charged Cyanine Fluorophores for Trafficking Scaffold Degradation. Biomed. Mater., 2013(8), 014109 (9pp). doi:10.1088/1748-6041/8/1/014109). However, these dyes have no reactive groups by which they can be covalently attached to hydrogel molecules. They are kept in the gelatin only due to weak hydrophobic interactions. As a result, these dyes can easily migrate from the gelatin. Moreover, due to these dyes having similar spectral and luminescence properties (intensity) in gelatin as in the free form they do not allow tracing of the hydrogel. In addition, the free dyes have a tendency to accumulate in the liver, lymph nodes and salivary glands.
Another method involves the in-vivo determination of PEG-dextran hydrogels and collagen using the covalently attached fluorescent dyes Texas Red and Fluorescein (N. Artzi et al. In vivo and in vitro Tracking of Erosion in Biodegradable Materials Using Non-invasive Fluorescence Imaging. Nat. Mater., 2011(10), 704-709).
The same approach is used in the non-invasive determination of chitosan membranes covalently labeled with tetrametyl rhodamine isothiocyanate (TRITC) (C. Cunha-Reis et al. Fluorescent Labeling of Chitosan for Use in Non-invasive Monitoring of Degradation in Tissue Engineering. J. Tissue Eng. Regen. Med., 2013 (7), 39-50). Collagen, covalently labeled with the cyanine dye ZW800-1 (S. H. Kim et al. Near-infrared Fluorescence Imaging for Noninvasive Trafficking of Scaffold Degradation. Sci. Rep. 2013 (3), 1198) and alginate hydrogel covalently labeled with Fluorescein isothiocyanate (FITC) (J. Liu et al. Synthesis, Characterization, and Application of Composite Alginate Microspheres with Magnetic and Fluorescent Functionalities. J. App. Polymer Sci. 2009 (113), 4042-4051; H. Zhu et al. Combined Physical and Chemical Immobilization of Glucose Oxidase in Alginate Microspheres Improves Stability of Encapsulation and Activity. Bioconj. Chem. 2005 (16), 1451-1458 and rhodamine B isothiocyanate (RITC). A method for the determination of a biodegradable hydrogel implant based on PEGylated fibrinogen (PF), covalently bound to the fluorescent cyanine dye Cy5.5 NHS ester (Cy5.5-NHS) was previously disclosed [Regenerative medicine applications in organ transplantation. Ed. G. Orlando, J. P. Lerut, S. Soker, R. J. Stratta, Elsevier, 2014, 452-453]. Hydrogel implants in form of cylindrical plugs, spherical micro-beads, or hydrogel precursors (chemicals, from which the hydrogel is generated by thickening or polymerization) are injected and polymerized in-situ, i.e. directly at the site of introduction. Cy5.5 provides a fluorescent signal for in-vivo determination and quantification of the resorption of the hydrogel and its degradation products, which are covalently bound to Cy5.5 as well as free Cy5.5 eliminated from the hydrogel during its degradation.
Covalent attachment of the dye molecules prevents their separation from the hydrogel molecules, but the dyes used are not sensitive to the environment, i.e. their spectral characteristics, such as the luminescence wavelength and/or intensity do not change upon changing the properties of the medium (viscosity, polarity and hydrophilicity). These methods therefore cannot differentiate between the non-degraded, dense hydrogel (e.g. hydrogel implant or carrier) with high viscosity and the lower-density hydrogel degradation products with low viscosity, and are not capable to detect the dense hydrogel. Moreover, the luminescence contribution of free dye eliminated from the dense hydrogel during the degradation process further makes it impossible to differentiate between the dense and degraded hydrogel regions. Therefore, these methods are incapable to determine rheological properties of a hydrogel (FIG. 1).
Importantly, luminescence based methods that allow differentiation between and/or localization of the high density hydrogels and their degradation products, e.g., by characterizing the rheological state of the hydrogel, have NOT been disclosed previously.